T cell receptors (TCRs) mediate recognition of a foreign or self-peptide presented in the context of a self major histocompatability (MHC) complex protein (1). During development, T cells expressing the highest affinity receptors are deleted in the thymus (negative selection). Survival of the T cell, however, depends on a minimal affinity for self MHC (positive selection), assuring that T cells recognition is MHC restricted (2). After development, if recognition of peptide:MHC (pepMHC) is of sufficient affinity, the T cell becomes activated and causes lysis of the target cell. The affinity of the TCR:pepMHC interaction is relatively low, with KD values on the order of 1-500 uM (3).
In recent years, in vitro engineering by yeast and phage display has yielded T cell receptors with >1,000 fold improvements in affinity (4-9). To date there have been only three MHC restricted human TCRs engineered for such improved affinity (reviewed in (10) and Varela-Rohena, 2008 (9). These three receptors were engineered in a full-length TCR format, with the addition of two non-native cysteines in each of the TCR constant domains (Ca and Cβ) to facilitate TCR heterodimer formation through an interchain disulfide bond (11). The addition of this disulfide bond allows the TCR to be expressed at higher levels on the surface of yeast (12) or phage (11). Other engineering efforts have focused on using a single-chain TCR (scTCR) format, consisting of the variable domains of the T cell receptor connected by a flexible linker (Vα-linker-Vβ or Vβ-linker Vα). Although the first scTCRs were described eighteen years ago (13, 14), there have been major difficulties in producing quantities sufficient for clinical or even pre-clinical use (10). Although analogous to antibody fragments in many aspects (reviewed in (10)), unlike the hundreds to thousands of antibody single-chain fragments (scFv) that have been expressed to date, the scTCR format has required mutations to allow for stable, higher level expression of the variable domains in the absence of the constant domains (12). Accordingly, stable scTCRs have been isolated by yeast display only through random mutagenesis and subsequent selection with clonotypic antibodies to select for mutations that allow for surface expression on yeast and expression at high levels in soluble form (6, 15, 16). These stable scTCR scaffolds were subsequently used for engineering receptors of enhanced affinity (4, 6).
Single-chain TCRs afford significant advantages in contrast to the full length TCR format for engineering, soluble protein expression, and clinical potential. From the perspective of further engineering, higher surface levels of the scTCR can be achieved relative to the full-length TCR in the yeast display system (12). As more TCR molecules are displayed on the surface of each yeast cell, there are improved avidity effects in the detection of peptide-MHC multimers and improved sensitivity of detection. From the perspective of soluble protein expression (i.e. manufacturing), the scTCR is produced as a single polypeptide, avoiding the requirement for production of each TCR chain as separate polypeptides and allowing for production of larger quantities of the properly assembled scTCR that binds to its peptide-MHC ligand. This feature can allow for production yields that are necessary for clinical use. Finally, from the clinical perspective, scTCRs can be formatted as therapeutics or diagnostic reagents similar to scFv fragments. The scTCR has the added advantage of improved tissue penetration because of the reduced size of the scTCR relative to the full-length receptor. This is especially important in cases when the TCR is directed against a tumor antigen, where tumor stroma can be targeted to effectively destroy cancerous cells (17).
Previous efforts to engineer single-chain TCRs were facilitated by having probes available to assess proper folding of both TCR V domains. In the well-characterized 2C (15) and 3L.2 (6) TCR systems, stabilized scTCRs were identified using clonotypic antibodies that recognized the conformation of the properly folded Vα and Vβ domains. Because the wild-type affinity of these receptors for specific pepMHC is relatively low, pep:MHC tetramers could not be used to detect properly folded, stabilized scTCRs.
In the present invention, the three known high-affinity human T cell receptors were cloned into a single-chain format to isolate the first human stabilized scTCRs. The features of the scTCRs that allowed them to be expressed as stable proteins are described. For example, the high-affinity scTCRs were used as templates for random mutagenesis (error-prone PCR), and the stabilized scTCRs were isolated by selection with soluble pep:MHC tetramers. Because the TCRs selected already contained mutations which enhance affinity for pep:MHC, soluble pep:MHC tetramers could be used as probes for the selection of scTCRs with stabilizing mutations. The scTCRs were subsequently produced in large quantities in E. coli. More specifically, the isolation of mutants of two stabilized, human scTCRs are described: 1) A6 scTCR specific for a peptide derived from the human T cell lymphotrophic virus Tax protein (peptide: Tax11-19, Sequence: LLFGYPVYV, SEQ ID NO:1), and 2) 868 scTCR specific for a human immunodeficiency virus derived peptide from the GAG protein (peptide: SL977-85, Sequence: SLYNTVATL, SEQ ID NO:2). Both of these TCRs used the same exceptionally stable Vadomain: Vα2 (IMGT: TRAV12 family, Table 1). Herein, it is also demonstrated that a single mutation in the Vα2 controls resistance to thermal denaturation of the Vα2 domain and enhances the stability of the corresponding Vβ domain of the scTCR. The scTCRs described herein have many applications. As an example of an application of the scTCRs of the invention for targeting the peptide-MHC antigens, we show the ability of the soluble 868 scTCR to recognize antigen presenting cells with a peptide derived from HIV Gag protein at low nM peptide concentrations. As a further example of an application of the scTCRs of the invention, the scTCR can be fused to a T cell signaling construct, allowing the gene transfected T cells to mediate recognition of the specific peptide-MHC and a response to the specific peptide-MHC.